This project has its final goal to understand the structural changes in tubulin related to nucleotide hydrolysis and resulting in the dynamic nature of microtubules. To this aim, we will compare the structures of the tubulin dimer in two different conformation representing the high and low energy states of the protein: the conformation in a straight protofilament where the tubulin-GDP molecule is constrained in a "GTP-like" state by the polymer lattice, and the conformation in rings where are curved protofilaments where tubulin-GDP is in a relaxed state. The structure of tubulin in the constrained state has been recently obtained in Downing's lab by electron crystallography of zinc-induced sheets. The present project will be concerned with obtaining the structure of the tubulin rings by cryo-electron microscopy of vitrified samples and image reconstruction using single particle methods. An immediate step will be to obtain a medium resolution (approximately 15 A) projection of the ring structure. The interpretation of the projection will be helped by comparison with the atomic model of the constrained tubulin in sheets. Some general differences between the two conformations of tubulin could already be gained at this stage, allowing us to test proposed hypothesis on the effect of nucleotide content on the structure of the tubulin dimer. The next steps will involves reconstructions of increasing resolution as the data collection and processing progresses. A reconstruction at 10 Angstroms will test us not only the overall change in the shape of the dimer, but whether there are any tertiary structure rearrangements in the molecule; which secondary structure elements are most affected by the nucleotide state; and how these structural changes may affect the assembly properties of tubulin.